In-vitro inhibition of IFNγ+ iTreg mediated by monoclonal antibodies against cell surface determinants essential for iTreg function

BMC Immunology

Gerhard Opelz¹  Haihao Wang²  Mahmoud Sadeghi¹  Volker Daniel¹ 

[1] Department of Transplantation-Immunology, Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, Heidelberg, 69120, Germany;Institute of Organ Transplantation, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, 430030, China

关键词: Cell proliferation;    Inhibition;    HLA-DR;    CD95;    CD28;    CD279;    CD152;    CD178;    IFNγ⁺CD127^-;    IFNγ⁺Foxp3⁺;    IFNγ⁺ iTreg;   

Others  :  1077883
DOI  :  10.1186/1471-2172-13-47

 received in 2012-02-09, accepted in 2012-08-16,  发布年份 2012

【 摘 要 】

Background

IFNγ-producing CD4⁺CD25⁺Foxp3⁺ PBL represent a subtype of iTreg that are associated with good long-term graft outcome in renal transplant recipients and suppress alloresponses in-vitro. To study the mechanism of immunosuppression, we attempted to block cell surface receptors and thereby inhibited the function of this iTreg subset in-vitro using monoclonal antibodies and recombinant proteins.

Methods

PBL of healthy control individuals were stimulated polyclonally in-vitro in the presence of monoclonal antibodies or recombinant proteins against/of CD178, CD152, CD279, CD28, CD95, and HLA-DR. Induction of IFNγ⁺ iTreg and proliferation of effector cells was determined using four-color fluorescence flow cytometry. Blockade of iTreg function was analyzed using polyclonally stimulated co-cultures with separated CD4⁺CD25⁺CD127^-IFNγ⁺ PBL.

Results

High monoclonal antibody concentrations inhibited the induction of CD4⁺CD25⁺Foxp3⁺IFNγ⁺ PBL (anti-CD152, anti-CD279, anti-CD95: p < 0.05) and CD4⁺CD25⁺CD127^-IFNγ⁺ PBL (anti-CD178, anti-CD152, anti-CD279, anti-CD95: p < 0.05). Effector cell proliferation increased with increasing antibody concentrations in culture medium (anti-CD178 and anti-CD279: p < 0.05). Conversely, high concentrations of recombinant proteins induced formation of CD4⁺CD25⁺Foxp3⁺IFNγ⁺ PBL (rCD152 and rCD95: p < 0.05) and decreased cell proliferation dose-dependently (rCD178 and rCD95: p < 0.05). Our data suggest an inverse association of iTreg induction with effector cell proliferation in cell culture which is dependent on the concentration of monoclonal antibodies against iTreg surface determinants. 3-day co-cultures of polyclonally stimulated PBL with separated CD4⁺CD25⁺CD127^-IFNγ⁺ PBL showed lower cell proliferation than co-cultures with CD4⁺CD25⁺CD127^-IFNγ^- PBL (p < 0.05). Cell proliferation increased strongly in CD4⁺CD25⁺CD127^-IFNγ^- PBL-containing co-cultures in the presence of monoclonal antibody (anti-CD28, anti-CD152, anti-CD279: p < 0.05) but remained low in co-cultures with CD4⁺CD25⁺CD127^-IFNγ⁺ PBL (with the exception anti-CD28 monoclonal antibody: p < 0.05). Monoclonal antibodies prevent iTreg induction in co-cultures with CD4⁺CD25⁺CD127^-IFNγ^- PBL but do not efficiently block suppressive iTreg function in co-cultures with CD4⁺CD25⁺CD127^-IFNγ⁺ PBL.

Conclusions

CD178, CD152, CD279, CD28, CD95, and HLA-DR determinants are important for induction and suppressive function of IFNγ⁺ iTreg.

【 授权许可】

   
2012 Daniel et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Daniel V, Naujokat C, Sadeghi M, Weimer R, Renner F, Yildiz S, Opelz G: Observational support for an immunoregulatory role of CD3 + CD4 + CD25 + IFN-gamma + blood lymphocytes in kidney transplant recipients with good long-term graft outcome. Transpl Int 2008, 21(7):646-660.
• [2]Daniel V, Sadeghi M, Wang H, Opelz G: CD4(+)CD25(+)Foxp3(+)IFN-γ(+) human induced T regulatory cells are induced by interferon-γ and suppress alloresponses nonspecifically. Hum Immunol 2011, 72(9):699-707.
• [3]Daniel V, Sadeghi M, Wang H, Opelz G: CD4+CD25+Foxp3+IFN-γ+ induced human Treg are a heterogenous cell population suppressing alloresponses specifically as well as unspecifically. Transpl Int 2011, 24(Suppl 2):114.
• [4]Daniel V, Sadeghi M, Wang H, Opelz G: CD4(+)CD25(+)Foxp3(+)IFNγ(+)CD178(+) human induced Treg (iTreg) contribute to suppression of alloresponses by apoptosis of responder cells.Hum Immunol. 2012 Sep 24. pii: S0198-8859(12)00561-7 doi:10.1016/j.humimm.2012.09.010. [Epub ahead of print].
• [5]Stroopinsky D, Avivi I, Rowe JM, Avigan D, Katz T: Allogeneic induced human FOXP3(+)IFN-gamma(+) T cells exhibit selective suppressive capacity. Eur J Immunol 2009, 39(10):2703-2715.
• [6]Feng T, Cao AT, Weaver CT, Elson CO, Cong Y: IL-12 converts Foxp3 + regulatory T cells to Foxp3 + IFN-gamma + T Cells with inhibitory functions during induction of colitis. Gastroenterology 2011, 140(7):2031-2043.
• [7]McClymont SA, Putnam AL, Lee MR, Esensten JH, Liu W, Hulme MA, Hoffmuller U, Baron U, Olek S, Bluestone JA, et al.: Plasticity of human regulatory T cells in healthy subjects and patients with type 1 diabetes. J Immunol 2011, 186(7):3918-3926.
• [8]Klein S, Kretz CC, Krammer PH, Kuhn A: CD127(low/-) and FoxP3(+) expression levels characterize different regulatory T-cell populations in human peripheral blood. J Invest Dermatol 2010, 130(2):492-499.
• [9]Semple K, Nguyen A, Yu Y, Wang H, Anasetti C, Yu XZ: Strong CD28 costimulation suppresses induction of regulatory T cells from naive precursors through Lck signaling. Blood 2011, 117(11):3096-3103.
• [10]Kolar P, Knieke K, Hegel JK, Quandt D, Burmester GR, Hoff H, Brunner-Weinzierl MC: CTLA-4 (CD152) controls homeostasis and suppressive capacity of regulatory T cells in mice. Arthritis Rheum 2009, 60(1):123-132.
• [11]Shen T, Zheng J, Liang H, Xu C, Chen X, Zhang T, Xu Q, Lu F: Characteristics and PD-1 expression of peripheral CD4 + CD127loCD25hiFoxP3+ Treg cells in chronic HCV infected-patients. Virol J 2011, 8:279. BioMed Central Full Text
• [12]Mkrtichyan M, Najjar YG, Raulfs EC, Abdalla MY, Samara R, Rotem-Yehudar R, Cook L, Khleif SN: Anti-PD-1 synergizes with cyclophosphamide to induce potent anti-tumor vaccine effects through novel mechanisms. Eur J Immunol 2011, 41(10):2977-2986.
• [13]Reardon C, Wang A, McKay DM: Transient local depletion of Foxp3+ regulatory T cells during recovery from colitis via Fas/Fas ligand-induced death. J Immunol 2008, 180(12):8316-8326.
• [14]Chen A, Liu S, Park D, Kang Y, Zheng G: Depleting intratumoral CD4 + CD25+ regulatory T cells via FasL protein transfer enhances the therapeutic efficacy of adoptive T cell transfer. Cancer Res 2007, 67(3):1291-1298.
• [15]Gritzapis AD, Voutsas IF, Lekka E, Papamichail M, Baxevanis CN: Peptide vaccination breaks tolerance to HER-2/neu by generating vaccine-specific FasL(+) CD4(+) T cells: first evidence for intratumor apoptotic regulatory T cells. Cancer Res 2010, 70(7):2686-2696.
• [16]Baatar D, Olkhanud P, Sumitomo K, Taub D, Gress R, Biragyn A: Human peripheral blood T regulatory cells (Tregs), functionally primed CCR4+ Tregs and unprimed CCR4- Tregs, regulate effector T cells using FasL. J Immunol 2007, 178(8):4891-4900.
• [17]Weiss EM, Schmidt A, Vobis D, Garbi N, Lahl K, Mayer CT, Sparwasser T, Ludwig A, Suri-Payer E, Oberle N, et al.: Foxp3-mediated suppression of CD95L expression confers resistance to activation-induced cell death in regulatory T cells. J Immunol 2011, 187(4):1684-1691.
• [18]Kisielewicz A, Schaier M, Schmitt E, Hug F, Haensch GM, Meuer S, Zeier M, Sohn C, Steinborn A: A distinct subset of HLA-DR+−regulatory T cells is involved in the induction of preterm labor during pregnancy and in the induction of organ rejection after transplantation. Clin Immunol 137(2):209-220.
• [19]Otsubo K, Kanegane H, Kamachi Y, Kobayashi I, Tsuge I, Imaizumi M, Sasahara Y, Hayakawa A, Nozu K, Iijima K, et al.: Identification of FOXP3-negative regulatory T-like (CD4(+)CD25(+)CD127(low)) cells in patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. Clin Immunol 2011, 141(1):111-120.
• [20]Feng G, Gao W, Strom TB, Oukka M, Francis RS, Wood KJ, Bushell A: Exogenous IFN-gamma ex vivo shapes the alloreactive T-cell repertoire by inhibition of Th17 responses and generation of functional Foxp3+ regulatory T cells. Eur J Immunol 2008, 38(9):2512-2527.
• [21]Feng G, Wood KJ, Bushell A: Interferon-gamma conditioning ex vivo generates CD25 + CD62L + Foxp3+ regulatory T cells that prevent allograft rejection: potential avenues for cellular therapy. Transplantation 2008, 86(4):578-589.
• [22]Warnecke G, Feng G, Goto R, Nadig SN, Francis R, Wood KJ, Bushell A: CD4+ regulatory T cells generated in vitro with IFN-{gamma} and allogeneic APC inhibit transplant arteriosclerosis. Am J Pathol 2010, 177(1):464-472.
• [23]Issa F, Chandrasekharan D, Wood KJ: Regulatory T cells as modulators of chronic allograft dysfunction. Curr Opin Immunol 2011, 23(5):648-654.